1. Field of the Invention
The present invention relates to prothrombin time reagents for monitoring extrinsic blood coagulation activities, a method for preparing the reagent and a method for using the reagent. More particularly, the invention relates to a liquid and dried synthetic prothrombin time reagents, prepared from recombinant tissue factor and synthetic phospholipids, which are stable for an extended time and can be dried under ambient conditions.
2. Description of Related Art
Blood coagulation tests may be performed for a variety of purposes, including determination of the bleeding susceptibility of patients undergoing surgery and monitoring of patients undergoing anti-coagulation therapy for the prevention of blood clots. A number of coagulation tests presently are in use. One of these tests is the xe2x80x9cprothrombin timexe2x80x9d (PT) test. The PT test relies upon the induction of the extrinsic coagulation protease factor VIIa by thromboplastin in a blood sample to be tested. The extrinsic coagulation pathway results in the production of thrombin, a proteolytic enzyme that catalyzes the conversion of fibrinogen to fibrin, which is essential to the clotting process.
Thromboplastin, also known as tissue factor, is a membrane associated glycoprotein that forms a complex with blood coagulation factor VIIa. The complex formed enhances the proteolytic activity of factor VIIa. The functional activity of prothrombin depends on the presence of phospholipids (Bach, Ronald R., Initiation of Coagulation by Tissue Factor, CRC Critical Reviews in Biochemistry 1988; 23(4): pp.339-368). Once formed, the factor VIIa/tissue factor complex activates a series of specific enzymes that comprise the extrinsic and common pathways of the coagulation cascade, which ultimately lead to the formation of thrombin, fibrin, platelet activation, and finally clot formation (Nemerson, Yale, Tissue Factor and Hemostasis, Blood 1988; 71: pp. 1-8).
The prothrombin time (PT) test utilizes this series of enzymatic events in vitro under controlled conditions to diagnose dysfunctions or deficiencies in the blood coagulation system of patients. Other uses include the monitoring of patients undergoing anticoagulant therapy. The time period it takes for clot formation to occur is the Prothrombin Time, or PT value.
PT reagents are used to monitor the coagulation activities of the extrinsic pathway of plasma, including those from patients on Coumadin(copyright) therapy. For the PT test, a highly sensitive reagent with an International Sensitivity Index (ISI) of 1.0 is desired. With an ISI of 1.0, the calculation of the International Normalized Ratio (INR) of PT coagulation test is simplified.
A PT reagent must have the following characteristics: sensitivity to abnormal samples, a well-defined normal PT value for normal plasma, providing accurate and reproducible results, maintaining consistency from lot to lot, and stability for storage either in a liquid state or in a dried state and upon reconstitution.
Tissues of vertebrates that have been added to citrated plasma and then recalcified accelerate clotting time. The tissue constituent that activates the coagulation protease cascade is commonly referred to as thromboplastin, or tissue factor (TF). Tissue factors employed in the present PT tests contain crude tissue factor extracted from natural sources. Natural sources include rabbit brain, rabbit brain/lung tissue mixtures, human placenta, or ox brain. Each of these sources has problems associated with them. For example, rabbit brain thromboplastin shows some seasonal variability, it varies from lot to lot, and is in relatively short supply. Human tissue factor may contain HIV or other viral diseases. Ox brain provides values that are much longer than those observed when employing tissue factor from alternative sources. Crude tissue factor preparations from natural sources also contain other coagulation factors as contaminants. The contamination with coagulation factors results in coagulation factor assay curves that are less sensitive to factor-deficient plasmas.
Tissue factor requires phospholipids for functional activity. Phospholipids found in PT reagents generally are those that adhere to tissue factor when it is extracted from animal sources. For example, the extraction of rabbit brain results in the concurrent isolation of both tissue factor and naturally occurring phospholipids which are bound to the tissue factor in vivo and survive the extraction process. No additional lipids are usually added to such extracted tissue factor. As a result, the nature, quantity, and quality of the lipids employed in the PT reagent will therefore vary depending upon the starting tissues and the extraction process, and will lead to lot to lot inconsistencies. The DADE(copyright) thromboplastin reagents, Thromboplastin C, C+, and IS, available from Dade International, Inc. of Deerfield, Ill., are all based on extracts of acetone-dehydrated rabbit brain. Partially purified extracts are blended with specific mixtures of buffers and stabilizers. Since the partially purified tissue factor extract is not completely delipidated, the addition of lipids back into the extract is unnecessary. The nature and composition of the resulting lipids is not well defined and can vary from lot to lot.
Different thromboplastin preparations either improve or reduce discrimination between blood samples having different prothrombin times. Thromboplastins with greater discrimination are termed xe2x80x9cmore sensitive.xe2x80x9d The liquid phase sensitivity of a preparation is graded by use of the ISI value. The value is found by plotting on a logarithmic scale, the prothrombin time seen with a thromboplastin lot in question versus the prothrombin time values seen with a standardized lot of thromboplastin. The ISI value is the slope of the resulting line multiplied by the ISI of the reference thromboplastin. More sensitive thromboplastins have lower ISI numbers around 1.0, and less sensitive thromboplastins have higher ISI numbers, typically around 2 to 3.
In attempting to avoid the problems associated with tissue factor from natural sources, the use of recombinant tissue factor for use in a PT reagent is described in U.S. Pat. No. 5,625,036 titled xe2x80x9cPreparation of Prothrombin Time Reagents from Recombinant Human Tissue Factor and purified Natural and Synthetic Phospholipidsxe2x80x9d issued Apr. 29, 1997 to P. L. Hawkins, et al., incorporated herein by reference. As described in the patent, human tissue factor is cloned and expressed in a number of organisms including E. coli. A portion of the cloned tissue factor is employed in the PT reagent without loss of functional activity, since most of the intracellular (cytoplasmic) domain of the cloned tissue factor can be truncated. The PT reagent includes recombinant tissue factor, phospholipids, either synthetic or natural, calcium ion, and a buffer composition. Well known cryopreservatives may also be added such as trehalose, maltose, and mannitol. Hawkins and other prior art preparation methods invariably require lyophilization or freeze-drying of the reagent for stability upon storage.
U.S. Pat. No. 5,314,695 titled xe2x80x9cTissue Factor Based Prothrombin Time Reagentxe2x80x9d issued May 24, 1994 to S. M. Brown, incorporated herein by reference, relates to a tissue factor prothrombin time reagent in which the tissue factor is inserted into the phospholipid bilayer of liposomes or phospholipid vesicles. A buffer containing a cryopreservative and glycine preferably forms part of the formulation. Either natural tissue factor or recombinant tissue factor can be used. The formulation composition is adjusted to allow maximum coagulant activity and sensitivity to extrinsic coagulation factors. Brown""s described preparation methods also require lyophilization.
U.S. Pat. No. 5,358,853 titled xe2x80x9cLiquid Thromboplastin Reagentxe2x80x9d issued Oct. 25, 1994 to J. R. Butler, et al., included herein by reference, describes a stable liquid thromboplastin reagent with a long shelf life which is prepared without lyophilization. However, the crude rabbit brain extract and unpurified phospholipids which comprise the liquid thromboplastin reagent of U.S. ""853 could cause variability of the reagent with regard to the PT test.
U.S. Pat. No. 5,418,141 titled xe2x80x9cTest Articles for Performing Dry Reagent Prothrombin Time Assaysxe2x80x9d issued May 23, 1995 to S. E. Zweig, et al., incorporated herein by reference, relates to test articles for performing dry reagent prothrombin time assays. The test articles comprise a solid phase membrane having dry, lyophilized thromboplastin immobilized therein. Coagulation neutral agents that facilitate rehydration of the dry thromboplastin are also provided on the solid phase membrane.
U.S. Pat. No. 5,508,170 titled xe2x80x9cLiquid Thromboplastin Reagentxe2x80x9d issued Apr. 16, 1996 to J. R. Butler, et al., incorporated herein by reference, relates to a stable liquid thromboplastin reagent which is derived from an acetone extract of rabbit brain tissue. The reagent described in U.S. Pat. No. 5,508,170 has a shelf life of greater than 16 months when packaged in a sealed container and is described as overcoming the inherent problems accompanying lyophilization, including: (1) variability in the filling of the vials before lyophilization; (2) shelf-to-shelf, and shelf positional differences in the lyophilization cycle (freezing and heating); (3) pipette errors associated with reconstitution and/or wrong volume additions when reconstituting the powder; and (4) water used to reconstitute may not be pure and/or may be contaminated with microorganisms. However the PT reagent sensitivity of the reagent described in U.S. Pat. No. 5,508,170 is substantially lower compared to recombinant tissue factor (e.g. approx. 2.0 Level III/I sensitivity compared to 4.0) because of the compositional variability of the rabbit brain tissue extract used in its preparation.
World Patent Application No. WO 98/48283 titled xe2x80x9cRecombinant Rabbit Tissue Factor Based Prothrombin Time Reagentxe2x80x9d published Oct. 29, 1998 to C. Brucato, et al., incorporated herein by reference, relates to liquid and lyophilized thromboplastin reagents derived from recombinant rabbit brain tissue factor where the removal of surfactant after mixing the surfactant-solubilized tissue factor with phospholipids is not necessary. However, no stability or shelf-life information with respect to the disclosed liquid thromboplastin reagent is provided.
Regarding another aspect of PT reagent formulation, a heterogeneous carbohydrate, Maltodextrin M-700 (a product of Grain Processing Corporation, Muscatine, Iowa), has been previously used (U.S. Pat. No. 5,591,403 titled xe2x80x9cPortable Prothrombin Time Test Apparatus and Associated Method of Performing a Prothrombin Time Testxe2x80x9d issued Jan. 7, 1997 to M. Gavin, et al., incorporated herein by reference). Maltodextrin and other polymeric carbohydrates are, in general, mixtures of several carbohydrates and may also contain aldehyde groups which could make an adduct with amino groups of proteins, such as tissue factor. Such heterogeneous carbohydrates may also be characterized by lot to lot variation, resulting in variation of performance of PT reagents formulated with such carbohydrates.
There remains a need therefore for a highly sensitive and consistent PT reagent that also remains stable either in the liquid stage or upon drying, but in either case without the complexity of lyophilization or impregnation into a membrane matrix, thereby rendering the PT reagent easier to manufacture, handle, transport, store, and use.
Briefly described, the invention comprises a PT reagent, a method of producing a PT reagent with a high degree of reproducibility for determining PT values, and a method of using the reagent. The inventive reagent is also highly stable upon either drying above 10xc2x0 C. in air, nitrogen, or a vacuum, or upon exposure to air at elevated temperatures in the liquid state. The inventive reagent has a long shelf life at 20xc2x0 C. in either the liquid or dried state, and remains stable at 37xc2x0 C. for about 2 weeks or longer.
Recombinant tissue factor, preferably containing a portion derived from rabbit brain, is solubilized and mixed with a mixture of solubilized, synthetic phospholipids in an appropriate ratio in the presence of one or more substituted or unsubstituted beta, gamma, or delta amino acids as stabilizing compounds. Suitable synthetic phospholipids include palmitoyloleoylphosphatidylcholine and palmitoyloleoylphosphatidylserine. Suitable amino acid stabilizing compounds include beta alanine, gamma aminobutyric, and delta aminovaleric acid, etc. Other useful stabilizing compounds are illustrated in Table A. In a preferred embodiment, the phospholipids are solubilized in a suitable detergent such as is octylglucoside dissolved in a suitable buffer such as Hepes buffer,(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) containing one or more of either beta alanine, gamma aminobutyric acid, or delta aminovaleric acid as the amino acid stabilizing compound. Other ancillary ingredients may be used, such as e.g., a salt such as sodium chloride(NaCl), an antioxidant such as BHT (butylated hydroxytoluene), a carrier protein such as bovine serum albumin (BSA), and a humectant such as sorbitol (D-glucitol). Upon solubilizing the components, the solution is subjected to dialysis.
A hydrophobic resin such as polystyrene divinylbenzene, polystyrene or polyacrylic resin, and the like, is optionally placed in the dialysis medium to increase dialysis efficiency. For example, Bio-Beads SM-2(copyright), a polystyrene divinyl benzene has been reported to be especially effective; see e.g. J. Philippot, et al., xe2x80x9cA Very Mild Method Allowing the Encapsulation of Very High Amounts of Macromolecules into Very Large (1000 nm) Unilamellar Lipsomesxe2x80x9d, Biochimica et Biophysica Acta; 734 137-143 (1983).
The dialysis medium comprises e.g. a buffer such as Hepes buffer, one or more of a beta, gamma, or delta amino acid stabilizing compounds, a salt such as sodium chloride, and a humectant such as sorbitol.
Upon completion of dialysis, the inventive composition is formulated and optionally dried above 10xc2x0 C. in air, nitrogen or a vacuum in a relative humidity above about 2% RH. Other combinations of temperature, gas composition, and humidity may be used provided that they allow the inventive composition to dry to the final desired moisture level, preferably about 2 to 10% moisture, in the desired time period. The dried materials are stored in a suitable container and are reconstituted with either water, plasma, blood, or the like; for use in coagulation-based assays when needed.
The liquid or reconstituted, air-dried reagent of the present invention is suitable for use in coagulation instruments such as the Fibrometer, MLA instrument of Medical Laboratory Automation Inc (Pleasantville, N.Y.), and the ACL instrument of Instrumentation Laboratories, Inc. (Lexington, Mass.).
The prothrombin time reagent and methods of the present invention employ recombinant protein tissue factor, one or more synthetic phospholipids comprising e.g. palmitoyloleoylphosphatidylcholine (POPC) and palmitoyloleoylphosphatidylserine (POPS) and one or more beta, gamma or delta amino acid stabilizing agents for monitoring the activity of the extrinsic blood coagulation pathway.
In the present invention, the recombinant tissue factor is preferably combined with or lipidated by a mixture of two synthetic phospholipids. Preferably, recombinant tissue factor derived from rabbit brain is employed. Recombinant tissue factor (r-TF) derived from rabbit brain is available from commercial sources. For example, in the present invention, a suitable r-TF derived from rabbit brain is available as a clear solution from Pel Freeze, Inc. of Rogers, Ark.; and is characterized as an apoprotein consisting of a fusion molecule of approx 45 Kd, containing extracellular and transmembrane domains of tissue factor, plus an amino terminal leader sequence consisting of several domains which aid in expression and purification. U.S. Pat. No. 5,858,724 titled xe2x80x9cRecombinant Rabbit Tissue Factorxe2x80x9d, issued Jan. 12, 1999 to R. E. Novy Jr., et al., contains a further description of a suitable r-TF and is hereby incorporated by reference. The tissue factor is solubilized in a suitable detergent, e.g. octylglucoside and then combined with or lipidated by a mixture of synthetic phospholipids. Preferably, the concentration of the r-TF for the reagent preparation is 200 to 250 xcexcg/ml.
Synthetic phospholipids, which are conveniently obtained from the manufacturer in ampoule form, are dissolved in a detergent solution and mixed together in a suitable ratio for the present composition. The synthetic phospholipid combination utilized in the present invention are not found in naturally occurring phospholipid mixtures. Preferably, the synthetic lipids employed are 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS), and the like. POPC and POPS are further characterized in that they contain one double bond in their molecular structures and their phase transition temperature are xe2x88x922 and 14xc2x0 C. respectively (See e.g. xe2x80x9cThermotropic Phase Transitions of Pure Lipids in Model Membranes and Their Modifications by Membrane Proteinsxe2x80x9d J. R. Silvius, Lipid-Protein Interactions, J. Wiley and Sons, Inc., New York, 1982; and Lipid Thermotropic Phase Transition Database (LIPIDAT)xe2x80x94NIST Standard Reference Database 34). In a preferred embodiment, POPC and POPS are added in a molar ratio range of about 6:4 to 8:2 respectively, preferably about 7:3. The preferred synthetic lipids have been previously described by J. H. Lawson and K. G. Mann, J. Biol., Chem., 266, 11317 (1991). The advantage of utilizing synthetic phospholipids as opposed to natural phospholipids is that the composition of natural phospholipids is variable, depending upon the source. In contrast, the chemical composition of synthetic phospholipids is more defined and therefore more consistent.
POPC and POPS are available from a number of commercial sources, including e.g. Avanti Corporation (Alabaster, Ala.), sold under Catalog No. 850457 and 830034, respectively. The synthetic phospholipids are dissolved in a suitable detergent solution such as octylglucoside. Preferably, the phospholipids dissolved in the mixture have a concentration in the range of about 11 to 14 mM preferably in the range of about 12 to 13 mM.
The tissue factor, along with a suitable carrier protein, such as bovine serum albumin, is lipidated by the dissolved phospholipids by solubilizing in an aqueous solution of a suitable detergent. Octylglucoside (n-Octyl xcex2-D-glucopyranoside) is preferred and is available from Sigma Chemical Co., Catalog No. O8001. Other detergents may be used provided the concentration of micelles or liposomes in the detergent is high enough to permit later dialysis of the solution. The concentration of the detergent, e.g. octylglucoside, is in the molar ratio of 15 to 65 mM with respect to the phospholipid, preferably in the molar ratio of 45 to 50 mM.
The amino acid stabilizer compound is represented by the following general formula I or a precursor thereof;
(I) H2Nxe2x80x94CR1R2xe2x80x94CR3R4xe2x80x94(CR5R6)nxe2x80x94(CR7R8)mxe2x80x94COOH;
wherein n represents 0 or 1; m represents 0 or 1; and
R1 and R2 may be the same or different and represent a hydrogen atom, a hydroxyl group, an amino group, an alkyl group, an alkoxyl group, a thioether group, an aryl group, or a heterocyclic group; either substituted or unsubstituted; and
R3, R4, R5, R6, R7, and R8 may be the same or different and represent a hydrogen atom, a hydroxyl group, an amino group, a carboxyl group, a carbonyl containing group, an alkyl group, an alkoxyl group, a thioether group, an aryl group, or a heterocyclic group; either substituted or unsubstituted.
Precursor compounds such as esters or salts are known in the art and are here defined as compounds that may form the inventive amino acid stabilizer compound in situ during the preparation or storage of the PT reagent. Such precursor compounds may be substituted in whole or in part for the free amino acid stabilizer compound described above.
Examples of suitable amino acid stabilizer compounds, also known as beta amino acids when n is 0 and m is 0, or gamma amino acids when n is 1 and m is 0, or delta amino acids when n is 1 and m is 1, are provided in Table A. Several inventive and comparative amino acid compounds were formulated to make PT reagents, and their effect on PT sensitivity (i.e. Level III/I ratio) was measured (see Examples 1 to 13 below). These experimental results are summarized in Table B. Satisfactory results mean that a Level III/I ratio of between 4.0 and 5.0 was obtained, while unsatisfactory results mean that a Level III/I ratio outside this range was obtained.
Beta alanine, gamma aminobutyric, and delta aminovaleric acid are especially preferred amino acid stabilizer compounds due to their low cost and ready availability. The inventive amino acid stabilizer compounds are added in the concentration range of about 0.1 to 10.0% (w/v), preferably in the range of about 1.0 to 5.0% (w/v), with an especially preferred range of about 1.5 to 2.5% (w/v). Suitable amino acid stabilizer compounds are also soluble or dispersible in the aqueous medium.
Suitable carrier proteins function as a stabilizer for in vitro reactions, especially involving proteins. Suitable carrier proteins include BSA, ovalbumin, and gamma globulin and the like, and are used in a concentration of about 0.01 to 1.0% (by weight). Preferably BSA is used at about 0.3% and may be obtained from Sigma Chemical Co. (St. Louis, Mo.).
The inventive solution also optionally contains a suitable antioxidant, especially if air-drying is employed to prepare the PT reagent. Antioxidants prevent e.g. the oxidation of lipids to fatty acids. Suitable antioxidants include butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), or Vitamin E and the like, in a concentration range of about 0.001 to 0.10% (by weight). Preferably, BHT is used at about 0.01% by weight.
In addition, the solution contains a suitable buffer such as Hepes, MOPS (4-Morpholinepropanesulfonic acid), TES (2-[[tris(hydroxymethyl)methyl]amino]-1-ethanesulfonic acid), DIPSO (3-[bis(2-hydroxyethyl)amino]-2-hydroxy-1-propanesulfonic acid), and the like, in a concentration range of 10 to 100 mM. Preferably the buffer used is Hepes at about 14 mM in concentration.
A salt is used such as sodium chloride, potassium chloride, and the like, in a concentration range of about 0.02 to 0.2 M. Sodium chloride is preferably used in a concentration amount of between about 30 mM and 60 mM.
A suitable amino acid chelating agent is optionally used such as L-serine, threonine, alpha alanine, and the like, in a concentration range of about 0.1 to 10% (by weight). Amino acid chelating agents can prevent precipitation by chelating metal ions that may be present in the composition as a result of the formulation process. Preferably L-serine is used at about 4 to 5% (by weight). Suitable amino acids are also employed during micelle or liposome formation along with recombinant rabbit brain tissue factor during the process of making the inventive composition such as beta alanine, glycine, alpha alanine, L-serine, and the like. Such amino acids are employed in a concentration of about 1 to 10% (by weight) with respect to the phospholipids. Preferably beta alanine or glycine is used in a concentration of about 4 to 6% (by weight).
One or more humectants are used such as glycerol (1,2,3-propanetriol), glycerol esters, sorbitol, ethylene glycol, propylene glycol, polyethylene glycol, and the like, in a total concentration range of about 1.0 to 10.0% (by weight). Preferably glycerol and sorbitol are used in a concentration range of about 0.1 to 0.5% (by weight) and about 2.0 to 7.0% (by weight), respectively when the invention reagent will be dried. A humectant, such as sorbitol, is added to the reagent mixture for stability and to provide tackiness or stickiness upon optional drying of the reagent. Although the mixture can be optionally dried to a powder form without a humectant, it has been found that the tackiness provided by a humectant is preferable when reconstituting the reagent. Moreover, the stickiness prevents the dried reagent from migrating within a test cuvette. Glycerol also imparts stickiness to the dried reagent, and is also preferably added as a moisture protectant to the solution when a dry reagent is desired.
An aldehyde free polymeric carbohydrate may be optionally used to adjust the sensitivity of the inventive PT reagent. Such a compound is defined as a polymeric carbohydrate which has no aldehyde group in its molecular structure. Preferably an aldehyde free synthetic polymeric carbohydrate is used which has a purity of greater than 90% (by weight) with respect to both molecular structure and molecular weight variation. Most preferably, gamma cyclodextrin (1297 MW) which contains 8D-glucopyranose units by alpha-(1-4) linkage is used in the inventive PT reagent; preferably at about 1% w/v in concentration on a diluted basis.
As discussed above, one or more synthetic lipids are dissolved in a suitable detergent solution and recombinant tissue factor is added followed by a carrier protein. The detergent can be removed by several methods known in the art to form a phospholipid tissue factor micelle (see e.g. L. T. Mimms, et al., xe2x80x9cPhospholipid Vesicle Formation and Transmembrane Protein Incorporation Using Octyl Glucosidexe2x80x9d, Biochemistry, 20, 833-840 (1981), and R. Bach, et al., xe2x80x9cFactor VII Bonding to Tissue Factor in Reconstituted Phospholipid Vesicles: Induction of Cooperativity by Phosphatidylserinexe2x80x9d, Biochemistry, 25, 4007-4020 (1986)). Suitable methods for detergent removal include dialysis, ultrafiltration, hydrophobic chromatography and the like.
In a preferred embodiment, the synthetic phospholipids, POPC and POPS, are dissolved in a detergent solution, in a molar ratio of 7 to 3 respectively, along with recombinant tissue factor and a beta or gamma amino acid stabilizer. The mixture is incubated at room temperature with gentle shaking for 60 minutes. The detergent in the mixture is then removed by dialysis.
In one preferred method, dialysis proceeds by placing the mixture in a dialysis bag and dialyzing at about 4xc2x0 C. against one or more Hepes buffer solutions. Dialyzed material in Hepes buffer also preferably contains 5% beta alanine as the amino acid stabilizer, 2% sorbitol and 0.1M sodium chloride. In another preferred embodiment, two dialysis solutions are used wherein the first solution contains 5% glycine and the second solution contains 5% L-serine as the respective amino acid chelating agents. After dialysis, the dialyzed material is drained from the dialysis bag and collected in a sterile polypropylene tube.
The dialyzed mixture containing the lipidated tissue factor is then diluted with a formulation buffer, preferably including Hepes, one or more beta, gamma or delta amino acid stabilizing compounds; sodium chloride, L-serine, sorbitol, BSA, BHT, a suitable biocide, and a suitable calcium ion source.
Suitable ionizable calcium sources include e.g. calcium salts of gluconate, acetate, or chloride and the like in a preferred concentration of range of about 10 to 20 mM. Calcium ions are essential for the activity of calcium dependent coagulation factors, such as Factors II, VII, IX and X. Calcium gluconate is preferred as the calcium ion source, and is preferably added in about a 14 mM concentration.
Suitable biocides include e.g. phenol, and antibiotics such as penicillin or Kanamycin and the like which are added in an effective concentration range to inhibit biogrowth in the inventive composition. Phenol in the concentration range of 0.1 to 0.2% (by weight) is preferred.
If a dried material is desired, the formulated reagent is dried without lyophilization at a temperature above about 10xc2x0 C. and a relative humidity of above about 2%. Preferably the material is air-dried at about 20xc2x0 C. and about 5% relative humidity, to yield a highly stable prothrombin time test reagent. Upon reconstitution with water, the reagent is subjected to coagulation assays employing a traditional prothrombin time test method, using a Fibrometer.
Concerning the ISI value, the inventive reagent provides a value of 1.0xc2x10.05 which greatly simplifies the calculation of the International Normalized Ratio (INR), as is apparent from the following formula for calculating the INR:
INR=(PT Ratio)ISI      PT    ⁢          xe2x80x83        ⁢    Ratio    =            PT      ⁢              xe2x80x83            ⁢      patient              mean      ⁢              xe2x80x83            ⁢      normal      ⁢              xe2x80x83            ⁢      PT      
The liquid or dried product of the present invention has an ISI value of substantially 1.0. An ISI value of 1.0 is preferred, since values reaching 2 or 3 indicate a less sensitive reagent.
The inventive reagent can be placed in a various coagulation instruments for testing. For example, the air-dried reagent is reconstituted and placed in a cuvette of the MLA instrument for assay purposes.
Thus, the highly sensitive reagent is optimized for long shelf life in either the liquid or dry state, without the need for lyophilization. If the material is dried, it is preferably air-dried under ambient conditions. Rehydration of the reagent is rapid, and the stickiness of the reagent prevents the reagent from moving within the chosen test device prior to reconstitution. The reagent is highly stable. The liquid or air-dried reagent, maintained at a temperature of 37xc2x0 C., remains stable for at least about 2 weeks.
Examples 1 to 12 which follow will serve to further typify the nature of the invention. Example 13 illustrates clotting time test results for comparative amino acids described in Table A. Examples 1 to 13 should not be construed as a limitation on the scope thereof, which is defined solely by the appended claims.
While the invention has been described with reference to the preferred embodiments thereof, it will be appreciated by those of ordinary skill in the art that modifications can be made to the parts that comprise the invention without departing from the spirit and scope thereof.